Toxicology Research, 2021, 10, 203–213 doi: 10.1093/toxres/tfaa106 Advance Access Publication Date: 15 February 2021 Review REVIEW A target safety assessment of the potential Downloaded from https://academic.oup.com/toxres/article/10/2/203/6135370 by guest on 30 September 2021 toxicological risks of targeting plasmepsin IX/X for the treatment of malaria Jane Barber,1 Phumzile Sikakana,1 Claire Sadler,1 Delphine Baud,2 ∗ Jean-Pierre Valentin3 and Ruth Roberts1,4, 1ApconiX, Alderley Park, Alderley Edge, SK10 4TG, UK, 2Medicines for Malaria Venture, 20 Route de Pré-Bois, Geneva 1215, Switzerland, 3UCB Biopharma SRL, Building R9, Chemin du Foriest, 1420 Braine-l’Alleud, Belgium and 4Biosciences, University of Birmingham, Edgbaston, B15 2TT, UK ∗ Correspondence address. ApconiX, Alderley Park, Alderley Edge, SK10 4DG, UK. Tel: +44 77 33 01 43 96; E-mail: [email protected] Abstract The aspartic proteases plasmepsin IX/X are important antimalarial drug targets due to their specificity to the malaria parasite and their vital role as mediators of disease progression. Focusing on parasite-specific targets where no human homologue exists reduces the possibility of on-target drug toxicity. However, there is a risk of toxicity driven by inadequate selectivity for plasmepsins IX/X in Plasmodium over related mammalian aspartic proteases. Of these, CatD/E may be of most toxicological relevance as CatD is a ubiquitous lysosomal enzyme present in most cell types and CatE is found in the gut and in erythrocytes, the clinically significant site of malarial infection. Based on mammalian aspartic protease physiology and adverse drug reactions (ADRs) to FDA-approved human immunodeficiency virus (HIV) aspartic protease inhibitors, we predicted several potential toxicities including β-cell and congenital abnormalities, hypotension, hypopigmentation, hyperlipidaemia, increased infection risk and respiratory, renal, gastrointestinal, dermatological, and other epithelial tissue toxicities. These ADRs to the HIV treatments are likely to be a result of host aspartic protease inhibition due a lack of specificity for the HIV protease; plasmepsins are much more closely related to human CatD than to HIV proteinase. Plasmepsin IX/X inhibition presents an opportunity to specifically target Plasmodium as an effective antimalarial treatment, providing adequate selectivity can be obtained. Potential plasmepsin IX/X inhibitors should be assayed for inhibitory activity against the main human aspartic proteases and particularly CatD/E. An investigative rodent study conducted early in drug discovery would serve as an initial risk assessment of the potential hazards identified. Received: 28 August 2020; Revised: 30 November 2020; Accepted: 7 December 2020 © The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 203 204 Toxicology Research, 2021, Vol. 10, No. 2 Graphical Abstract Downloaded from https://academic.oup.com/toxres/article/10/2/203/6135370 by guest on 30 September 2021 Introduction mitigation strategy. Here we explore the benefit–risk profile for a potential new target in treating malaria and propose next steps Despite the increase in drug approvals in recent years, drug to evaluate risk. discovery and development remain challenging with high rates of failure [1]. In the hunt for ways to make drug discovery and Malaria: the problem development more successful, there have been several excellent analyses of why drugs fail [2, 3]. Although the reasons for failure Malaria remains an overwhelming problem, particularly in are usually complex and multifactorial, ‘safety/toxicity’ is cited Africa. In 2018, there were an estimated 228 million cases of as the predominant cause of failure [2, 3] with around 40% of malaria with an estimated 405 000 deaths from the disease these failures attributable to safety issues associated with the globally. The burden of disease is heaviest in the World Health therapeutic target itself [2]. This is not surprising since many tar- Organization (WHO) African Region where 93% of all malaria gets that are attractive in treating disease are also likely to have deaths occurred. Plasmodium falciparum (P. falciparum) is the important roles in normal biology and their modulation could most prevalent malaria parasite (99.7%) in the WHO African lead to unintended consequence. So, would not it make sense to Region carried by the Anopheles mosquito, whereas P. vivax is the maximize our understanding of the biology of a potential drug predominant parasite (75%) in the WHO Region of the Americas. target in order to predict and manage issues before they arise? Children aged under 5 years old and pregnant women are in the For many projects, the drug target may be expressed in tissues most vulnerable groups affected by malaria; children aged under other than the intended therapeutic target; a careful analysis 5 account for >60% of malaria deaths in 2018 worldwide [4]. of expression profiles can help predict likely consequences of Classical uncomplicated malaria presents with a combination this and offer an insight into the benefit–risk profile. For most of symptoms including fever, chills, sweats, headaches, body anti-infectives (antibiotics, antifungals, antibacterials, antiproto- aches, nausea, and vomiting. Severe malarial infections are zoals), the target may be specific to the infectious agent suggest- characterized by serious organ failures or abnormalities in the ing that the potential for on-target toxicity is limited. However, patient’s blood or metabolism [5]. The current fight against this is not always the case since there may be related mammalian the disease is being waged on a variety of fronts, including targets that need to be considered to develop an overall risk the distribution of bed nets, the promotion of indoor spraying, Barber et al. 205 and the development of new medicines, vaccines, and insec- the cleavage of proteins that are to be exported to the host cell ticides. Emerging parasite resistance to the currently available [18], and is therefore already a focus of drug development. PMIX drugs is of increasing major concern. and PMX have recently been highlighted as mediators of egress The first instance of antimalarial drug resistance was with and invasion of the parasite further exposing the parasite at its chloroquine. Chloroquine resistance in P. falciparum was devel- most vulnerable state accentuating these enzymes as potential oped independently in multiple areas of Southeast Asia, Oceania, drug targets [15, 19, 20]. and South America in the 1950/60s. Subsequently chloroquine Within the erythrocyte the Plasmodium parasite matures resistance spread to nearly all areas of the world where P. fal- from a ring to a trophozoite and then a schizont which ciparum malaria is transmitted [6]. Since then P. falciparum also produces merozoites primed for invasion (Fig. 1). The release developed resistance to nearly all of the other currently avail- of merozoites, known as egress, is a two-step process: the able antimalarial drugs, including sulfadoxine/pyrimethamine, degradation of the parasitophorous vacuole (PV), which encloses mefloquine, halofantrine, and quinine [7]. Although resistance the merozoites, and erythrocyte membrane. Invasion of a new to these drugs tends to be much less widespread geographically, erythrocyte takes only 10–30 s and involves recognition of the in some areas of the world, the impact of multidrug resistant erythrocyte membrane, attachment, reorientation, and entry Downloaded from https://academic.oup.com/toxres/article/10/2/203/6135370 by guest on 30 September 2021 malaria can be extensive [8]. Most recently, resistance to the [21]. The proteins involved in invasion and egress are packaged artemisinin and non-artemisinin components of artemisinin- into several secretory organelles in the merozoite including the based combination therapy has emerged in parts of Southeast rhoptries and micronemes (invasion) and the dense granule-like Asia, impacting the efficacy of this vital antimalarial class [9]. exonemes (egress). The activity of several serine and cysteine Chloroquine-resistant P. vivax malaria has also been identified proteases promotes the destabilization of the PV and erythrocyte in a number of regions including Papua New Guinea, Southeast membranes which surround the parasite [22]. PMX processes Asia, Ethiopia, and Madagascar [6]. Therefore, there is still a subtilisin-like protease 1 (SUB1) to activate it. Inhibition of PMX continuous need for novel, differentiated approaches to treat results in the accumulation of SUB1 precursor [10]. Mature SUB1 malaria. is required for the degradation of both the PV and erythrocyte membrane to allow the dissemination of merozoites from a Plasmepsins: drug targets for malaria mother schizont. To initiate the egress cascade, SUB1 activates cysteine proteases called SERAs and merozoite surface proteins As malaria routinely develops resistance to drugs, there is a con- called MSPs [23]. Full block of PMX traps parasites within the tinual need for novel antimalarials to combat the disease. This PV membrane, whereas partial block allows egress from this is especially vital in the case of multidrug resistant P. falciparum membrane but prevents escape from the erythrocyte membrane. as the number of available therapies are reducing and becoming This suggests a higher level of activated SUB1 is required for less effective. Development of small molecule plasmepsin (PM) its